CN112480223B

CN112480223B - Housefly derived antibacterial peptide D-26M and preparation method and application thereof

Info

Publication number: CN112480223B
Application number: CN202011155731.5A
Authority: CN
Inventors: 孔德龙
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2020-10-26
Filing date: 2020-10-26
Publication date: 2023-04-07
Anticipated expiration: 2040-10-26
Also published as: CN112480223A

Abstract

The invention discloses a housefly derived antibacterial peptide D-26M and a preparation method and application thereof, wherein the housefly antifungal peptide MAF-1A is subjected to amino acid residue substitution and dextrorotation modification to obtain D-26M, and the sequence of the D-26M is shown in a sequence table SEQ IQ No. 2; and (3) performing solid-phase synthesis on the polypeptide by using a polypeptide synthesizer, purifying by using reverse high performance liquid chromatography, and finally performing amino acid analysis and mass spectrum identification. The invention improves the stability and cell selectivity of D-26M under the condition of ensuring high antibacterial activity; the D-26M has stronger cationic property, hydrophobicity and capability of penetrating into a pathogenic bacteria double-layer membrane, has broad-spectrum antibacterial activity and efficient anti-inflammatory activity, particularly has good resistance to multi-drug-resistant acinetobacter baumannii, free acinetobacter baumannii in-vivo and in-vitro environments and a biofilm formed by the acinetobacter baumannii, and can effectively inhibit the activity of outer membrane LPS of gram-negative bacteria.

Description

Housefly derived antibacterial peptide D-26M and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, particularly relates to an antibacterial peptide, a preparation method and application thereof, and particularly relates to a housefly derived antibacterial peptide D-26M, a preparation method and application thereof.

Background

According to the 'antibacterial drug review' published in 2016, 70 million people die of infection caused by antibiotic-resistant pathogens every year at present, and 1000 million people face the life risk every year by 2050 if a solution for slowing down the occurrence of drug resistance cannot be found. The separation rate and the drug resistance rate of acinetobacter baumannii in clinical specimens are increased year by year, which are the main pathogenic bacteria causing nosocomial infection; endotoxin (LPS) released in the process of gram-negative bacterial infection continuously stimulates an immune system of an organism, induces endotoxin shock, septicemia, urinary infection, nosocomial acquired pneumonia and the like caused by pathogenic bacteria infection, and has a mortality rate of up to 52% especially in Intensive Care Units (ICUs). In addition, acinetobacter baumannii can continuously exist in the external environment, and the pathogenic bacteria seriously harm public health, so that the acinetobacter baumannii becomes a problem to be solved urgently. Studies have now shown that acinetobacter baumannii has a resistance rate to tigecycline of up to 66% and carbapenem antibiotics, which are considered to be the best means for the clinical treatment of drug-resistant bacterial infections, of 46-66%, whereas acute kidney damage occurs in about half of all patients receiving polymyxin treatment of acinetobacter baumannii infections. Therefore, a need exists for new antibacterial agents against acinetobacter baumannii infection in clinic, and the world health organization also ranks the development of new agents against acinetobacter baumannii as the most superior/critical pathogen.

Antimicrobial peptiDes (AMPs) are small molecule polypeptides that an organism can resist infection by an external pathogen. Compared with the traditional antibiotics, AMPs have the characteristics of wide antibacterial spectrum, unique antibacterial mechanism and difficult generation of drug resistance, have great potential for developing novel antibacterial drugs, and are very promising to replace the traditional antibiotics for clinical treatment. However, AMPs have the disadvantages of poor stability, low activity and the like, thereby limiting the clinical application of AMPs. Therefore, according to the known factors capable of influencing the bioactivity of AMPs, the AMPs molecular modification and the efficient peptide screening are carried out to overcome the defects of AMPs, and the method becomes an important research field for the development of peptide antibacterial drugs. Important factors affecting the biological activity of AMPs include the amino acid composition of AMPs, the peptide chain length, the net charge amount, the α -helicity, and the like. Housefly (Musca Domestica) is a vector insect distributed worldwide, and due to the complex structure and diversity of pathogenic microorganisms in the breeding environment, the high-efficiency disease resistance of the housefly taking antibacterial peptide as an effective molecule is widely concerned.

Disclosure of Invention

The invention aims to provide a housefly derived antibacterial peptide D-26M, which can improve the stability and cell selectivity of AMPs while keeping high antibacterial activity.

In order to realize the aim, the housefly derived antibacterial peptide D-26M has a sequence shown in a sequence table SEQ ID No. 2.

Preferably, the antimicrobial peptide D-26M has an alpha-helical structure.

Another objective of the invention is to provide a preparation method of the housefly derived antibacterial peptide D-26M, the preparation method is low in cost, and the prepared derived antibacterial peptide D-26M has stronger cationic property, hydrophobicity and capability of permeating into a pathogen double-layer membrane.

In order to realize the aim, the preparation method of the housefly derived antibacterial peptide D-26M comprises the following steps:

(1) Performing amino acid sequence mutation and dextrorotation modification on the polypeptide MAF-1A by adopting an amino acid residue substitution method, wherein the sequence of the polypeptide MAF-1A is shown as a sequence table SEQ IQ No. 1; the specific process is as follows: replacing Glu and Asp in the amino acid sequence of the polypeptide MAF-1A with dextrorotatory Lys; replacing Gln and Lys at position 23 in the amino acid sequence of the polypeptide MAF-1A by dextral Leu; replacing Lys at positions 9 and 15 in the amino acid sequence of the novel peptide MAF-1A with D-Trp containing indole side chain; the rest amino acids in the polypeptide MAF-1A are replaced by corresponding dextrorotatory amino acids; obtaining a derivative antibacterial peptide D-26M with the length of 26, wherein the sequence of the derivative antibacterial peptide D-26M is shown in a sequence table SEQ IQ No. 2;

(2) Adopting a solid phase chemical synthesis method, and synthesizing the derivative antibacterial peptide by a polypeptide synthesizer;

(3) And purifying the synthesized derivative antibacterial peptide by using reverse high performance liquid chromatography, and performing amino acid analysis and mass spectrum identification on the polypeptide after purification to finish the preparation of the derivative antibacterial peptide.

The invention also aims to provide the application of the housefly derived antibacterial peptide D-26M in preparing a bacteriostatic agent for treating and/or preventing pathogenic bacteria.

Preferably, the pathogenic bacteria include escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, staphylococcus aureus and bacillus subtilis.

Preferably, the pathogenic bacterium is acinetobacter baumannii.

The invention also aims to provide the application of the housefly derived antibacterial peptide D-26M in preparing a medicament for treating and/or preventing inflammatory diseases caused by bacterial endotoxin LPS.

Another objective of the invention is to provide application of the housefly derived antibacterial peptide D-26M in preparing medicines for treating and/or preventing various nosocomial and extranosocomial infectious diseases and inflammatory diseases.

Preferably, the various nosocomial and extranosocomial infectious and inflammatory diseases include pneumonia, septicemia, otitis media, meningitis, skin soft tissue infections, urinary system infections.

Compared with the prior art, the invention improves the stability and the cell selectivity of the derived antibacterial peptide D-26M under the condition of ensuring the high antibacterial activity of AMPs. The antibacterial peptide has stronger cationic property, hydrophobicity and capability of penetrating into a pathogen double-layer membrane, has broad-spectrum antibacterial activity and high-efficiency anti-inflammatory activity, has good bactericidal function on Escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, staphylococcus aureus, bacillus subtilis and the like, particularly has good resistance on multiple drug resistance Acinetobacter baumannii, free Acinetobacter baumannii in-vivo and in-vitro environments and a formed biofilm thereof, can effectively inhibit the activity of outer membrane LPS of gram-negative bacteria, and has good application prospect in developing traditional antibiotic alternative drug molecules for clinical anti-infectious and anti-inflammatory diseases. The antibacterial peptide consists of 26 amino acids, has a short peptide chain and is low in preparation cost.

Drawings

FIG. 1 is a spiral diagram of the template polypeptide MAF-1A and D-26M: (A) MAF-1A; (B) D-26M;

FIG. 2 is a time-kill curve of antimicrobial peptide D-26M against Acinetobacter baumannii: (A) short time 1h; (B) a long time of 12h;

FIG. 3 shows the scanning electron microscope results of the antibacterial peptide D-26M after acting on Acinetobacter baumannii: (A) a PBS negative control group; (B) D-26M test group; (C) a polymyxin B positive control group;

FIG. 4 shows the effect of antimicrobial peptide D-26M on Acinetobacter baumannii biofilm formation: (A) Determining the influence of different concentrations of D-26M and polymyxin B on the formation of the acinetobacter baumannii biofilm by crystal violet staining; (B) Statistical analysis of the amount of acinetobacter baumannii biofilm formation at different concentrations of D-26M and polymyxin B, { P } <0.001;

FIG. 5 is a graph showing the survival of C.elegans under the action of antimicrobial peptide D-26M at various concentrations;

FIG. 6 is a graph showing the effect of antimicrobial peptide D-26M on the infection of A.baumannii with C.elegans: (A) A pattern of in vivo bacterial load of the infected nematodes after different interventions; (B) survival profiles of infected nematodes after different interventions;

FIG. 7 shows the antagonistic effect of the antimicrobial peptide D-26M on LPS;

FIG. 8 is a graph showing the effect of antimicrobial peptide D-26M on LPS-induced macrophage inflammatory factor IL-1 β and TNF- α expression: (A) ELISA detection of IL-1 β cytokine secretion; (B) ELISA was performed to detect the secretion of TNF-. Alpha.cytokines.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples.

1. Experimental materials and methods

1. Materials:

strains and insect strains: escherichia coli ATCC25922, klebsiella pneumoniae ATCC700603, pseudomonas aeruginosa ATCC27853, staphylococcus aureus ATCC25923, bacillus subtilis ATCC6633 and Candida albicans ATCC10231 were purchased from American type culture collections repository; multiple drug resistant acinetobacter baumannii ATCC19606 was purchased from the parasitology research laboratory of the medical university of guizhou (isolated from the national hospital of the province of guizhou), and the wild type caenorhabditis elegans (WT Bristol N2) was purchased from the genetics laboratory of the medical university of xuzhou.

Drugs and reagents: MHB medium, LB medium, sandcastle glucose broth (SDA), trypsin, and agar powder were purchased from beijing solibao corporation; RPMI-1640 medium was purchased from G, USA; crystal violet dye liquors were purchased from Dalian Meilun Co Ltd; polymyxin B, BHI medium, tryptone soy medium were purchased from Beijing Soilebao GmbH; the CCK8 kit was purchased from gangrenes bio ltd; microplate quantitative matrix staining method limulus reagent was purchased from building door limulus reagent ltd; mouse IL-1. Beta. ELISAKit and Mouse TNF-. Alpha.ELISA Kit were purchased from Thermo corporation; trizol reagent was purchased from Invitrogen; other analytical reagents were purchased from Beijing Biyuntian BioLimited.

The main apparatus is as follows: cliniBio 128Ce microplate reader from beton instruments ltd, usa; 5804R high speed refrigerated centrifuge is available from Eppendorf, germany; scanning electron microscopy was purchased from FEI corporation, usa; the real-time fluorescent quantitative PCR system is purchased from Roche, switzerland; 043BR1549 protein electrophoresis apparatus is available from Bio-RaD company, USA.

2. The method comprises the following steps:

modification and synthesis of derivative antibacterial peptide: based on the influence factors of cationic property, hydrophobic property, alpha-helical structure and the like, separating antifungal peptide MAF-1 (GENBANK: HM 178948) from Musca domestica larva haemolymph, intercepting 26 amino acids at 128-153 sites of the C end of the antifungal peptide to form polypeptide MAF-1A, carrying out amino acid sequence mutation and dextrorotation (D) transformation on the polypeptide MAF-1A by adopting an amino acid residue substitution method, and substituting dextrorotation Lys for Glu and Asp in the amino acid sequence of the MAF-1A in order to enhance the cationic property of the derivative peptide; to increase the hydrophobicity of the derivatized peptide and to increase the proportion of the alpha-helical structure, gln and Lys at position 23 in the MAF-1A amino acid sequence are replaced by D-Leu; to increase the capacity of AMPs to penetrate into the bilayer membrane of pathogenic bacteria, lys at positions 9 and 15 in the MAF-1A amino acid sequence was replaced by d-Trp containing indole side chain; the rest amino acids in the MAF-1A are replaced by corresponding dextrorotatory amino acids; introducing D-amino acid for modification, enhancing the stability of the derived peptide, designing the derived peptide with non-perfect amphipathy by combining bioinformatics analysis, naming the derived peptide as D-26M (the amino acid sequence is KKFKKTAKWLIKKLSKLLALLKMK, both are dextrorotatory amino acids), and analyzing the physicochemical properties of the derived peptide by utilizing ExPASY website software ProtParam (http:// web. Expasy.org/ProtParam /); helix mapping was performed using Heliquest software (http:// helix. Ipmc. Cnrs. Fr/cgi-bin/ComputParams. Py); the designed derivative antibacterial peptide is synthesized by adopting a solid-phase synthesis technology, is purified by reversed-phase high-performance liquid chromatography (RP-HPLC) (the purity is more than 97 percent), and is subjected to amino acid analysis and mass spectrum (ESI-MS) identification after being purified.

Analysis of antibacterial activity of the derivative antibacterial peptide: candida albicans, staphylococcus aureus, bacillus subtilis, escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii were inoculated on the plate, respectively, to prepare a bacterial suspension, with reference to the judgment standards of the liquid dilution method recommended by the American society for Clinical and Laboratory Standards (CLSI). The derivative antibacterial peptide D-26M is diluted by times of the culture medium and then is respectively added into the prepared bacterial suspensions. And incubating in an incubator at 37 ℃ for 24h, and measuring the absorbance at 600nm by using a CliniBio 128Ce microplate reader to obtain the Minimum Inhibitory Concentration (MIC) of the minimum concentration for inhibiting the growth of microorganisms in the bacterial suspension. Each antibacterial peptide sample is provided with 3 repeats, a sterile culture medium is used as a negative control, polymyxin B is used as a drug control, and a normal growth group is used as a positive control.

And (3) analyzing the antibacterial stability of the derived antibacterial peptide on acinetobacter baumannii: respectively using 150mmol/L NaCl, 4.5mmol/L KCl and 1mmol/L MgCl ₂ 、2.5mmol/L CaCl ₂ 0.15mg/ml Trypin and 0.6mg/ml Trypin solution are diluted to obtain derivative peptide D-26M in a multiple ratio, the final reaction concentration is sequentially 1.024mg/ml, 0.512mg/ml, 0.256mg/ml, 0.128mg/ml and 0.064mg/ml, and the change of the MIC value of D-26M to acinetobacter baumannii in different environments is measured by taking MAF-1A as a control.

Analysis of antibacterial time-bactericidal effect of the derivative antibacterial peptide on acinetobacter baumannii: preparing a bacterial solution with 0.5 McLeod concentration, uniformly mixing the Acinetobacter baumannii bacterial solution with derivative antimicrobial peptide D-M26 (the final concentration of the reaction is the MIC value of bacteria), shaking and culturing at 37 ℃, respectively diluting the bacterial solutions of 0h, 5min, 10min, 15min, 20min, 40min, 60min, 2h, 4h, 6h, 8h, 10h and 12h after culture, marking and coating plates, and counting bacterial colonies after culturing at 37 ℃ for 24 h. Sterile PBS is set as a negative control group, polymyxin B is set as a positive control group, and each group is subjected to repeated experiments for 3 times. And (3) taking different sampling times as an abscissa, taking logarithm lg (cfu/ml) of the number of the corresponding colonies of each time point as an ordinate, and drawing a time-sterilization curve.

The influence of the derived antibacterial peptide on the thallus is observed by a scanning electron microscope: taking Acinetobacter baumannii in logarithmic growth phase, and diluting the Acinetobacter baumannii to 1.0X 10 by using MHB culture medium ⁶ CFU/ml, using 1 × MIC antibacterial derived peptide as an experimental group, PBS as a control group and polymyxin B as a drug control group, placing the experimental group, PBS as a drug control group in a shaker at 37 ℃, shaking and culturing for 1h, centrifuging at low temperature for 10min at 2000r/min, discarding the supernatant, washing with sterile water for 3 times, and fixing with 2% paraformaldehyde and 3% glutaraldehyde overnight. 0.1 percent of the total amount of the raw materials is washed by PBS for 3 times, 1 to 2 percent of osmium tetroxide is immersed for 1 hour for dyeing, rinsed for 3 times by distilled water, dehydrated, dried, stuck on a table and plated by an S-3400N scanning electron microscope for detection in sequence of 70 percent ethanol, 80 percent ethanol, 90 percent ethanol and absolute ethanol.

And (3) analyzing the activity of the derivative antibacterial peptide on the Acinetobacter baumannii biofilm: the derivative peptide was diluted with the prepared bacterial solution to have a final concentration of 1/4 XMIC, 1/2 XMIC, MIC in this order. PBS is used as a negative control group, polymyxin B is used as a positive control group, and the constant temperature culture is carried out for 24 hours at 37 ℃; slowly washing with PBS for 3 times, and adding 99% methanol for fixation for 15min; adding 0.1% crystal violet dye solution for 5min, washing with sterile double distilled water, and air drying; adding 95% ethanol, standing on a shaking table at room temperature for 30min, measuring absorbance at 600nm with a microplate reader, and repeating each experiment for 3 times, wherein each concentration is 3 holes.

Evaluation of in vivo antibacterial Activity of derivatized antimicrobial peptides in caenorhabditis elegans infection model

Toxicity assay of the derivatized antimicrobial peptides against caenorhabditis elegans: culturing the nematodes to an L3 stage, washing the nematodes with an M9 buffer solution, and transferring the nematodes onto a sterile NGM solid culture dish; the prepared nematodes were picked up in BHI medium containing different concentrations of derivative antimicrobial peptides (8 × MIC, 4 × MIC, 2 × MIC, MIC) to contain about 25 nematodes per well, and a group containing no antimicrobial peptide was set as a growth control group. Culturing at 20 deg.C, observing and recording the death and survival number of nematodes by stereomicroscope every 24h, and continuously counting for 7 days.

C, establishing a caenorhabditis elegans-acinetobacter baumannii infection model and analyzing the antibacterial peptide action:

(1) Survival time analysis of acinetobacter baumannii-infected nematodes after antibacterial peptide action: coating acinetobacter baumannii on trypticase soy agar medium, and culturing at constant temperature of 37 ℃ for 18h; culturing the synchronized nematodes to an L3 stage, transferring the nematodes to the culture medium after M9 buffer solution is resuspended, infecting the nematodes for 6 hours at 25 ℃, and collecting nematode bodies; culturing at constant temperature of 25 ℃, observing and recording survival and death numbers of nematodes every 24 hours, and continuously observing for 7 days.

(2) Analyzing the number of bacteria in vivo after the acinetobacter baumannii-infected nematodes are subjected to the action of antibacterial peptide: respectively setting high-concentration (4 × MIC), medium-concentration (2 × MIC) and low-concentration (MIC) antibacterial peptide treatment groups; infecting and collecting the nematode, culturing at 25 deg.C for 24 hr, washing the nematode with M9 buffer solution for three times, and grinding; after the grinding fluid is diluted properly, the coated plate is placed in a constant-temperature incubator at 37 ℃ for incubation for 24h, and then counting is carried out.

Limulus test to detect neutralization of LPS by the derived peptide: the binding of the antimicrobial peptide to LPS was detected according to the instructions of the commercial Limulus test bacterial LPS detection kit, and the binding rate was counted as a percentage change from the untreated sample.

Analyzing the influence of the derivative antibacterial peptide on the expression of the LPS-induced proinflammatory factors: mouse RAW264.7 macrophage cells were cultured by the conventional method, and the derivative antimicrobial peptide with final concentration MIC was added for 1h in advance according to the following ratio of 1. ELISA is used for detecting the IL-1 beta and TNF-alpha content of the supernatant.

2. Results of the experiment

Properties of the polypeptide: the amino acid sequence and the characteristics of the modified full-dextrorotation polypeptide are shown in table 1, and the molecular weight of the modified full-dextrorotation polypeptide is 3103Da which is slightly increased compared with the parent peptide; the positive charge is +9, the hydrophobic amino acid accounts for 53.85%, the alpha-helix accounts for 50%, and the alpha-helix is slightly reduced compared with the parent peptide, and finally, a derivative peptide D-26M with a non-perfect amphiphilic structure is obtained, as shown in figure 1, the light-color background is the hydrophobic amino acid; the dark background is hydrophilic amino acid; the dextrorotatory amino acid substitution positions are marked with circles.

Properties of the polypeptide of Table 1

Note: d-amino acids are italicized.

Analysis of antibacterial activity of the derivative antibacterial peptide: the result is shown in table 2, compared with the template peptide MAF-1A, the modified derivative antibacterial peptide D-26M has the advantages that the antibacterial activity is obviously improved, the antibacterial spectrum is expanded, the gram negative bacteria effect is more obvious, and the antibacterial effect is close to the antibacterial level of the clinical traditional antibiotic polymyxin B.

TABLE 2 MIC determination of polypeptides against different pathogenic bacteria

"-": not measured

Stability test of the derived antibacterial peptide in salt ion and enzyme environments: antibacterial activity of D-26M in different ionic and enzymatic environmentsAs shown in Table 3, the ion concentration was measured in different ionic environments (150 mM NaCl, 4.5mM KCl, 1mM MgCl) ₂ 、2.5mM CaCl ₂ ) In the case of 150mM NaCl, the MIC value was not affected at all, but only at 4.5mM KCl, 1mmol/L MgCl ₂ And 2.5mmol/L CaCl ₂ Under the condition, the MIC value is increased by 1 time, the fluctuation is not large, and therefore, the MIC value of D-26M to the acinetobacter baumannii is hardly influenced. In the environment of different concentrations of pancreatin (0.15 mg/ml Trypsin and 0.6mg/ml Trypsin), the MIC value of D-26M on Acinetobacter baumannii was not affected. The D-26M shows that the inhibiting effect on the acinetobacter baumannii in the physiological environment has good stability.

TABLE 3 MIC value detection of D-26M for Acinetobacter baumannii under different physiological environments

And (3) determining the antibacterial time-bactericidal effect of the derivative antibacterial peptide on the acinetobacter baumannii: dynamically monitoring the bactericidal effect of the derivative antibacterial peptide D-M26 on the acinetobacter baumannii in a short time (1 h) and a long time (12 h) under the MIC concentration, and the result is shown in figure 2, wherein the acinetobacter baumannii in a normal control group without the drug normally grows; under the MIC concentration, D-26M acts for 10min and starts to kill acinetobacter baumannii, and the speed is obviously faster than that of positive control polymyxin B; meanwhile, the D-26M has the same long-acting bacteriostatic effect as the polymyxin B in 12 h.

The influence of the derived antibacterial peptide on the thallus is observed by a scanning electron microscope: and observing the shape change of the acinetobacter baumannii after different treatments are carried out for 1 hour by a scanning electron microscope. The results are shown in FIG. 3: the PBS negative control group (A) acinetobacter baumannii has full thallus, normal shape and smooth and flat surface; after Acinetobacter baumannii is treated by D-26M with MIC concentration (B), thalli is disintegrated, contents are released, the thalli loses the original cell structure, and the cell membrane is smoother and holes are formed only in partial positions by positive control polymyxin B treatment group (C). Thus, D-26M shows a bactericidal effect by destroying the cell membrane of Acinetobacter baumannii, and the membrane breaking effect is stronger than that of polymyxin B.

Effect of derivatized antimicrobial peptides on acinetobacter baumannii biofilms: the crystal violet staining results are shown in FIG. 4 (A), and when 1/4 XMIC, 1/2 XMIC, polypeptide with MIC concentration or polymyxin B as a positive control drug exist, the bacterial biofilm stained in dark color becomes gradually lighter; statistical analysis in FIG. 4 (B) shows that biofilm formation amounts of Acinetobacter baumannii treated at three concentrations of D-26M are 69.27%, 42.34% and 20.66% on average. The above results indicate that the higher the concentration of polypeptide in the environment, the less bacterial biofilm formation.

Toxicity analysis of the derived antimicrobial peptides against caenorhabditis elegans: as shown in FIG. 5, the survival rate of normally bred C.elegans after 7 days of culture is approximately 90% (C.elegans:)

Group); when the nematode culture environment contains D-26M with the concentrations of MIC, 2 XMIC, 4 XMIC and 8 XMIC, the survival curve of the nematode is not different from that of the group without the antibacterial peptide treatment. Indicating that the polypeptide is not toxic to nematodes at the concentrations tested.

Effect of derivatized antimicrobial peptides on acinetobacter baumannii infection of c.elegans: polypeptide intervention with high (4 × MIC), medium (2 × MIC) and low (MIC) concentrations is respectively given to caenorhabditis elegans infected with Acinetobacter baumannii, and as a result, as shown in figure 6 (A), the bacterial carrying capacity of nematodes in the infected group is obviously reduced after the polypeptide treatment, meanwhile, all nematodes in the infected group die after 2 days of infection, and the polypeptide can remarkably prolong the survival time of the infected nematodes, and the prolonged time are dose-dependent on the concentration, as shown in figure 6 (B). The result shows that the derived antibacterial peptide has good treatment effect on the acinetobacter baumannii infection.

Effect of derivatized antimicrobial peptides on bacterial endotoxin LPS: the activity of LPS after 1h of mixing with the polypeptide at MIC, 2 × MIC concentrations was only 61.42%, 47.97% of the LPS group, as shown in fig. 7, as a result of 1h incubation of the polypeptide at MIC, 2 × MIC concentrations with 0.25EU of LPS and relative activity of LPS detected using limulus reagent. It was shown that the polypeptide was able to affect the LPS activity and that higher concentrations had a greater effect on LPS activity.

Effect of derivatized antimicrobial peptides on LPS-induced inflammatory responses: as shown in the ELISA results in FIG. 8, D-26M significantly inhibited the ability of LPS to induce secretion of IL-1 β and TNF- α, which are macrophage inflammatory factors of RAW 264.7.

The experimental results prove that the modified derivative antibacterial peptide D-26M has broad-spectrum antibacterial activity and particularly has strongest antibacterial activity on drug-resistant acinetobacter baumannii. It is effective against gram-negative bacteria: the minimum inhibitory growth concentrations (MIC) of Escherichia coli ATCC25922, klebsiella pneumoniae ATCC700603, pseudomonas aeruginosa ATCC27853 and Acinetobacter baumannii ATCC19606 are respectively 2.5. Mu.M, 5. Mu.M and 2.5. Mu.M; MIC values of gram-positive bacteria staphylococcus aureus ATCC25923 and bacillus subtilis ATCC6633 are respectively 80 mu M and 20 mu M; the MIC value for the fungus Candida albicans ATCC10231 was 40. Mu.M. Therefore, the antibacterial peptide D-26M provided by the invention can be applied to medicines for treating or preventing pathogenic bacteria, particularly gram-negative bacteria, and the stability of the antibacterial peptide D-26M under different physiological environments is obviously improved compared with that of a template peptide.

The antibacterial mechanism research shows that D-26M has the advantage of quickly and durably killing acinetobacter baumannii, can sterilize after 10min of action and can effectively maintain for 12h, and is obviously faster than clinical medicine polymyxin B. The inhibition effect on the formation of bacterial biofilm is remarkable; the D-26M has low toxicity and stable antibacterial activity in vivo as found by using caenorhabditis elegans as an in vivo experimental model animal model. Meanwhile, D-26M can effectively antagonize the activity of bacterial endotoxin LPS and play a role in inhibiting inflammatory reaction induced by LPS. The D-26M provided by the invention has good in vivo and in vitro anti-Acinetobacter baumannii activity and anti-LPS activity, can be applied to medicaments for treating diseases caused by Acinetobacter baumannii infection, can also be applied to medicaments for treating or preventing inflammatory diseases caused by bacterial endotoxin LPS, and has good application prospects for various nosocomial and external infectious diseases and inflammatory diseases such as pneumonia, septicemia, otitis media, meningitis, skin soft tissue infection, urinary system infection and the like.

Sequence listing

<110> Xuzhou university of medicine

<120> housefly derived antibacterial peptide D-26M, preparation method and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Lys Lys Phe Lys Glu Thr Ala Asp Lys Leu Ile Glu Ser Ala Lys Gln

1 5 10 15

Gln Leu Glu Ser Leu Ala Lys Glu Met Lys

20 25

<210> 2

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Lys Lys Phe Lys Lys Thr Ala Lys Trp Leu Ile Lys Ser Ala Trp Leu

1 5 10 15

Leu Leu Lys Ser Leu Ala Leu Lys Met Lys

20 25

Claims

1. Housefly derived antibacterial peptideDThe preparation method of the-26M is characterized by comprising the following specific steps:

(1) Performing amino acid sequence mutation and dextrorotation modification on the polypeptide MAF-1A by adopting an amino acid residue substitution method, wherein the sequence of the polypeptide MAF-1A is shown in a sequence table SEQ ID No. 1; the specific process is as follows: replacing Glu and Asp in the amino acid sequence of the polypeptide MAF-1A by dextrorotatory Lys; replacing Gln and Lys at position 23 in the amino acid sequence of the polypeptide MAF-1A by dextro-Leu; replacing Lys at positions 9 and 15 in the amino acid sequence of the polypeptide MAF-1A by dextro-Trp containing indole side chain; the rest amino acids in the polypeptide MAF-1A are replaced by corresponding dextrorotatory amino acids; obtaining a derived antimicrobial peptide of length 26 D-26M, the sequence of which is shown in the sequence table SEQ ID No. 2;

2. The housefly-derived antibacterial peptide according to claim 1D-26M housefly derived antibacterial peptide prepared by preparation methodD-26M in preparation of bacteriostatic agent for treating and/or preventing pathogenic bacteria infection, and housefly derived antibacterial peptideDThe sequence of-26M is shown in a sequence table SEQ ID No.2, and is characterized in that: the pathogenic bacteria comprise Escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, acinetobacter baumannii, staphylococcus aureus and Bacillus subtilis.

3. The housefly derived antibacterial peptide according to claim 1D-26M housefly derived antibacterial peptide prepared by preparation methodD-26M in the preparation of bacteriostatic agents for the treatment and/or prevention of pathogenic bacterial infections, said housefly derived antimicrobial peptidesDThe sequence of-26M is shown in a sequence table SEQ ID No.2, and is characterized in that: the pathogenic bacteria are acinetobacter baumannii.

4. The housefly-derived antibacterial peptide according to claim 1D-26M housefly derived antibacterial peptide prepared by preparation methodD-26M in the preparation of medicaments for treating and/or preventing inflammatory diseases caused by bacterial endotoxin LPS, and the housefly derived antibacterial peptideDThe sequence of-26M is shown in the sequence table SEQ ID No. 2.